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Investigation Into the Etiology of Sudden Death of Cattle/livestock All Over the Bangladesh.

M. H. Al-Faruque
Animal Health Research Division,Bangladesh Livestock Research Institute, Savar, Dhaka 1341.

M. Parvin
Department of Pathology,Bangladesh Agricultural University, Mymensingh-2202

M. A. H. N. A. Khan
Department of Pathology,Bangladesh Agricultural University, Mymensingh-2202

P. Monura
Animal Health Research Division,Bangladesh Livestock Research Institute, Savar, Dhaka 1341.

M. Giasuddin
Animal Health Research Division,Bangladesh Livestock Research Institute, Savar, Dhaka 1341.

M. J. F. A. Taimur
Animal Health Research Division,Bangladesh Livestock Research Institute, Savar, Dhaka 1341.

Abstract/ Executive Summary:

The aim of this study was to identify the causes of sudden or unusual death of cattle and molecular diagnosis of foot and mouth disease virus. For identification of causes of death of cattle samples were collected from different parts of Bangladesh where unusual death of cattle occurred. Relevent history, presenting signs and required samples were collected from the affected animals for diagnosis. It was found that Foot and Mouth Disease (FMD) caused highest mortality (40.63%) followed by nitrate toxicity (15.63%), Black quarter (9.38%), anaplasmosis (6.25%), dermatophilosis (6.25%), aspiration pneumonia (6.25%), tetanus (3.12%), hypomagnesemic tetany (3.12%), acute ruminal acidosis (3.12%) and others (6.25%) respectively. In case of molecular diagnosis of FMD virus vesicular fluids were collected from clinically affected cattle. Then the sample mixed with buffered glycerine kept at -20% until use. Primers for general FMDV detection (P33/P32;IF/IR) and for sub typing 8 different Oligonucletide was synthesized by Riaken® Japan. Total RNA were extracted by qiagen minispin RNA easy kit. RNA was extracted from the stored epithelial fluid according to the manufacturer’s protocol. The RNA was resuspended in 50 µl RNase free water and 5 µl taken for reverse transcription. A commercially available random hexanucletide mix (Promega®) was used for first strand synthesis. RT-PCR and multiplex PCR was done according to protocol by Reid et Al., 2000 and Vangrysperre and De Clereq, 1996. The PCR product was examined by electrophoresis in 2% agarose gel containing 2.5 µg ethidium bromides and visualized under ultraviolet light. The expected sizes of PCR amplified fragments were 131bp and 328bp. Positive and negative controls were included in each assay to determine the reactivity of the RT and amplications steps. Finally, it was found that FMD virus and its different sub-types could be detected with RT-PCR method efficiently.

Keywords: Cattle, Unusual death, FMD, PCR.

Location: Bangladesh Livestock Research Institute, Savar, Dhaka 1341.

Start Date: 01-07- 2009

End Date: 30-06-2010

Program Area: Animal Health and Management

Commodity: Cattle

Objectives:

1. To identify the causes of unknown death in cattle. 2. To find out the remedies for the unknown death of cattle.

Methodology:

Outbreak investigation of cattle diseases in Bangladesh 1. Examination of sick animals: The emergency medical team moved to the site death of cattle with necessary equipment and preparation. The sick animals was examined in situ blood, urine, tissues (biopsy), stool was collected for routine for generating possible causes of cattle death. 2.Systemic dissection (necropsy) of dead animals: Samples were collected from the suspected animals or dead animals after getting information from related people. A routine post mortem examination was performed to detect possible caused of death. 3. Collection and shipment of samples: Samples were collected from the clinically sick or death animals. Animals like blood, urine, biopsy (lymph node, other suspected organs), rumen ingesta, fecal materials, plant materials etc were collected. These were transferred to the laboratory. Then the samples were processed in the laboratory for bacterial, viral, protozoal, fungal, nutritional or poisoning causes of death. Serum samples was analyzed in autoanalyzer for the detection of serum enzyme marker for the damage of specific organs. 4. Diagnosis of diseases: Presumptive diagnosis was done on the basis of the history, clinical signs, postmortem examination and results of routine blood and urine tests. Laboratory tests were performed in connection with the history and clinical findings. Grams staining, modified Wrights staining, histopathology, fecal examination and bacterial culture tests were performed both in the mobile laboratory and laboratories in the BAU/ BLRI. 5. Reverse Transcription-Polymerase Chain Reaction (RT-PCR): RT-PCR was used to detect the organisms. Detection of viral RNA in samples by RT-PCR involved three steps: isolation of RNA, RT-PCR and analysis of RT-PCR products by electrophoresis. 6. Sample preparation: Sample (Vesicle of foot) was collected aseptically into PBS in screw-capped test tube and stored at -70?. Then centrifuged at 5000 rpm for 15 minutes. Supernatant was collected in fresh sterile tubes and stored at -70?. 7. Isolation of RNA: Total RNA was extracted in Bangladesh Livestock Research Institute, Savar, Dhaka using Qiagen RNeasy kit. The RNeasy technology combines the selective bindings properties of a silica-gel-based membrane with the speed of microspin technology. High-salt buffer (Guanidine isothiocyanate, GITC) system allows up to 100g of RNA longer than 200 bases to bind to the RNeasy silica-gel membrane. Contaminants are efficiently washed away and high quality RNA is then eluted in 50’1 of Rnase free water. 8. Selection and synthesis of primers for RT-PCR: Two pairs of oligonucleotic primer were used to detect the foot and Mouth Disease virus Primers for FMDV were designed by comparison of published sequences. The tube containing RT-PCR products were kept out from the thermocycler and stored at 4? in refrigerator until electrophoresis. 9. Electrophoresis: RT-PCR products were analyzed by electrophoresis on 1.5% agarose gel, stained with ethidium bromide and examined under UV light using an image documentation system. 1. Gel casting tray was assembled with gel comb of appropriate teeth size and number. 2. 1.8% agarose solution was prepared in TAE buffer by melting in a microwave oven. 5 µl ethidium bromide (10mg/ml) was added to 100ml molten agarose to have a final concentration of .5 µl/ml. 3. Molten agarose was poured on to the casting tray and allowed to solidity on the bench. 4. The hardened gel in its tray was transferred to the electrophoresis tank containing sufficient TAE buffer to cover the gel ~1mm . The comb was gently removed. 5. 10 µl of each RT-PCR product was mixed with 2-3 µl loading buffer and the sample was loaded to the appropriate well of the gel. 6. 3 µl DNA size marker (100bp) was loadedin one well. 7. The leads of the electrophoresis apparatus were connected to the power supply and the electrophoresis was run at 92V. 8. When DNA migratefd sufficiently, as judge from the migration of bromophenol blue of loading buffer, the power supply was switched off. 9. The gel was gently washed in running water and placed on the transilluminator in the dark chamber of the image documentation system. The UV light of the system was switched on; the image was viewed on the monitor, focused, acquired and saved in a floppy disc, as well as printed on thermal paper.

Source of Information: BLRI. Proceedings of the Annual Research Review Workshop 2009-2010, Bangladesh Livestock Research Institute, Savar, Dhaka 1341.

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The expected sizes of PCR amplified fragments were 131bp and 328bp. Positive and negative controls were included in each assay to determine the reactivity of the RT and amplications steps. Finally, it was found that FMD virus and its different sub-types could be detected with RT-PCR method efficiently.

Knowledge Management: Report/Proceedings